Detection of amniotic fluid in vaginal secretions of pregnant women due to premature rupture of fetal membranes

ABSTRACT

A method is taught for the accurate determination of the premature rupture of membranes (PROM), defined as spontaneous rupture of membranes before the onset of uterine contractions. More specifically, a lateral flow assay strip tests for at least two antigens to greatly limit or eliminate the possibility of false negatives. A built in timer in the cassette holding the lateral flow assay further increases the accuracy of the test. A collection buffer vial with self-contained shipping and dropper caps and built in stand is also taught.

Premature rupture of membranes (PROM), defined as spontaneous rupture ofmembranes before the onset of uterine contractions, is one of the mostcommon diagnostic dilemmas in contemporary obstetric practice. PROM canoccur at any gestational age, and preterm PROM (PPROM, defined as PROMbefore 37 weeks) is responsible for 20-40% of preterm births. This mayresult in infections, fetal distress and preterm birth, responsible forperinatal morbidity and mortality, strong damage or disorders for thefetus. The treatment of PROM depends not only on the stage of pregnancyand on the fetus maturity but also on the risk of infection and fetaldistress. It is thus crucial to be able to diagnose PROM with accuracyat an early stage in order to decrease the risks of complications.(Michel et al., Electrophoresis 2006, 27, 1159-1181, incorporated hereinby reference).

Early and accurate diagnosis of PROM would allow for gestationalage-specific obstetric interventions designed to optimize perinataloutcome and minimize serious complications such as cord prolapse,preterm delivery, fetal distress and infectious morbidity(chorioamnionitis, neonatal sepsis). Conversely, a false-positivediagnosis of PROM may lead to unnecessary obstetric interventions,including hospitalization, administration of antibiotics andcorticosteroids, and even induction of labor. Therefore, the correct andtimely diagnosis of this disorder is of critical importance to theclinician because PROM and PPROM may be associated with serious maternaland neonatal consequences.

The comparison of the protein composition of samples from differenttissues or different physiological states is one of the possibilitiesthat can be used for the identification of PROM. Indeed, differentmethods based on clinical evaluation and biological tests have beendeveloped to diagnose PROM, among which the detection of proteins suchas prolactin, alpha-fetoprotein, diamine oxidase, insulin-like growthfactor binding protein (IGFBP-1), human chlorologonadotrophin, fetalfibronectin, cytokines, interleukin 6, interleukin 18, as well asdecidual relaxins H1 and H2 proteins, usually determined byimmunoassays. One of the problems encountered with these proteins isthat even if they are present in amniotic fluid (AF) at concentrationshigher than those in the maternal plasma, the contamination of a vaginalsection sample by maternal blood can lead to false-positive results.Moreover, no definite agreement on which marker to use has yet beenachieved, and none of these markers has really been accepted yet as thereference analyte by clinicians. Michel, supra.

Several studies presenting proteomic analyses of amniotic fluid andcells have been published which identify new potential markers for PROM,i.e., proteins present only in amniotic fluid and absent in maternalblood. Two peptides, corresponding to the COOH-terminus fragments ofagrin (Swiss-Prot O00468) and perlecan (Swiss-Prot P98160) have beenshown to be present only in amniotic fluid at the 17^(th) week ofpregnancy and have been considered as new potential makers of PROM.Other markers found present in the amniotic fluid at the 17^(th) week ofpregnancy include alkaline phosphatase placental like, IGFBP-1, perlecanand pregnancy associated endometrical alpha2 globulin.

Insulin-like growth factor-1 (IGFBP-1) is particularly important inhuman female reproductive physiology, where it is involved with otherfactors in a complex system which regulates menstrual cycles, puberty,ovulation, decidualization, implantation and fetal growth. This hasimplications for clinical obstetrics and gynecology, where there isevidence for a pathophysiological role for IGFBP-1 in pre-eclampsia,intrauterine growth restriction, polycystic ovarian syndrome andtrophoblast and endometrial neoplasms. IGFBP-1 was first purified fromplacenta and fetal membranes in 1980 (Bohn et al., Isolierung andCharacterisierung eines Neuen Plazentaspezifischen Proteins (PP12),Arch. gynecol. 1980; 229: 279-291). It was thought to be a protein ofplacental origin, and it was called Placental Protein 12 (PP12). Laterit was observed that PP12 and IGFBP-1 purified from amniotic fluid havethe same N-terminal amino acid sequence (Povoa et al., Cross-reactionsof serum somatomedin-binding protein in a radioimmunoassay developed forsomatomedin-binding protein isolated from human amniotic fluid. ActaEndocrinologica 1984; 107: 563-570) and that PP12 binds IGF I(Insulin-like Growth Factor I). However, it is not known for certainthat there are minor differences between the two protein molecules suchas conformational changes, etc.

The concentration of IGFBP-1 in amniotic fluid has been observed to beusually 100 to 1000 times higher than that in maternal serum (Rutanen etal., Radioimmunoassay of placental protein 12: levels in amniotic fluid,cord blood and serum of healthy adults, pregnant women and patients withtrophoblastic disease. Am. J. Obstet. Gynecol. 1982; 144: 460).Mammalian alpha-fetoprotein (AFP) is a single-chain glyprotein with amolecular mass ranging from 66 to 72 kD, synthesized by the fetal liver,gut and yolk sac. After entering the fetal urine it is readily detectedin the amniotic fluid. The concentration between fetal plasma AFP andmaternal serum AFP is approximately 150 to 200 fold. (Shahin, et al.,Comparative Study of 3 Amniotic Fluid Markers in Premature Rupture ofMembranes . . . , Gynecologic and Obstetric Investigation. 2007;63:195-199). Reactions based on specific binding substances aregenerally known. Antibodies are the compounds most commonly used asspecific binding substances. These so-called immunological methods arebased on the ability of an antibody to bind specifically to a certainsite in its antigen (epitope). The so-called polyclonal antibodies are amixture of immunoglobulins in serum of an immunized animal. The mixturesare different in each individual animal. Unlike this, the so-calledmonoclonal antibodies are produced by one cell line that is culturedunder laboratory conditions, and each antibody is homogenous and can becharacterized by methods used in protein chemistry and continuouslyproduced in identical form.

Rutanen applies methods which allow the total amount of IGFBP-1 in theblood and the amniotic fluid to be determined using MAb 6303 and MAb6305 In particular, Rutanen uses a method known as a two-siteimmunoradiometric assay which is described by F. Pekonen, et al., inJournal of Immunoassay, 1989, Vol. 10, pp. 325-337. This method consistsof placing a vaginal secretion sample into a sample-holding platecontaining one of the two above antibodies. The IGFBP-1 moleculecontained in the vaginal secretion sample is attached to the antibodywhich is present in the holding plate. An “antibody” here is a substancecapable of binding to a predetermined site of an antigen. An antigen isany substance eliciting an immunologic response such as the productionof an antibody specific for that substance.

Towards that end, there have been attempts to produce a test for thedetection of PROM.

U.S. Pat. No. 5,554,504 (Rutanen) discloses a diagnostic method fordetecting the rupture of fetal membranes. A diagnostic method fordetecting rupture of fetal membranes in a pregnant woman comprising:obtaining a sample of vaginal secretions from the pregnant woman,reacting the sample with at least one binding substance thatspecifically binds to insulin-like growth factor binding protein 1,IGFBP-1, in order to detect a level of IGFBP-1 in the sample, anddetecting a level of IGFBP-1 in the sample above a predeterminedthreshold level, the predetermined threshold level being selected asindicative of amniotic fluid in the vaginal secretion sample, wherein alevel of IGFBP-1 in the sample above the predetermined threshold levelis indicative of rupture of fetal membranes in the pregnant woman. Theantibody used is a monoclonal antibody specific for IGFBP-1.

Accurate diagnosis of fetal membrane rupture, however, remains afrequent clinical problem in obstetrics. Unfortunately, a non-invasivediagnostic gold standard is not available at this time. Currentlyavailable tests are inaccurate and require an intrusive examination. Thecurrent diagnostic methods using nitrazine/pH, assessment of pooling andmicroscopic fern testing lack specificity, become progressively lessaccurate with passage of time since membrane rupture are associated withhigh false-positive rates related to infection, presence of urine, bloodor semen. Buyukbayrak et al, discloses the use of vaginal washing-fluidprolactin level determination as an alternative diagnostic method forPROM. Omu et al, and Seyei et al, (U.S. Pat. No. 5,096,830) discloses amethod for determining increased risk of fetal membrane rupture byobtaining a secretion sample from the vaginal cavity to determine thepresence of a fetal antigen fetal fibronectin.

SUMMARY OF THE DISCLOSURE

The present disclosure uses a mixture of antibodies specificallyreactive with two or more amniotic fluid proteins to detect a fetalmembrane rupture. The antibodies are positioned on a lateral flow assaytest strip. The use of this method reduces the level of false negativesfrom more than 10% to virtually zero.

In one embodiment of the disclosure, monoclonal and polyclonalantibodies specific for certain amniotic fluid proteins are used.

In one embodiment of the disclosure, a liquid sample is obtained byplacing a long handled swab into the patient's vaginal vault. Thissample is then placed into a buffer solution, contained within a vial.

In another embodiment of the disclosure, the vial contains a stand, sothat there is less of a possibility of the vial being accidentallyknocked over. Additionally, a built in cap further reduces the chance ofthe sample spilling during shipping and handling.

In another embodiment of the disclosure, the swab is scored so that thestick of the swab can be easily broken off and left in the buffer vialfor better sample dispersion in the buffer and providing easier usewithout having to find proper disposal of the swab tip.

After the swab has had enough time to release the markers into thebuffer, the buffer sample is placed on the lateral flow assay in aspecific site, such that the antigens are tagged by colorant taggednon-specific antibodies. The fluid sample then migrates by capillaryaction to the site of the monoclonal and polyclonal antibodies, whereinthe antibodies specifically bind to the specific antigen present in thesample.

In one embodiment of the disclosure, the lateral flow assay test stripfits in, and is surrounded by a cassette.

In another embodiment of the disclosure, the lateral flow assay cassettecontains a timer showing the elapsed time of the test as an integralpart of the cassette.

The lateral flow assay test detects a combination of specific proteinspresent in amniotic fluid of pregnant women in all trimesters ofpregnancy. These specific proteins are unique and found in much higherconcentrations in amniotic fluid; therefore, they can be used asspecific markers for the diagnosis for the rupture of fetal membranes.

In another embodiment of the disclosure, the present tests are easier touse, and less cumbersome, than prior tests.

In one embodiment of the disclosure, both the (PP12) and betaIg-H3biomarkers may be used to detect a fetal membrane rupture.

In another embodiment of the disclosure, fetal fibrnectin may be used todetect fetal membrane rupture.

In another embodiment of the disclosure alpha-fetoprotein (AFP) andprolactin may be used to detect fetal membrane rupture.

In yet another embodiment, the detection of high hCG levels in vaginalfluids may be used in combination with the above mentioned

In one embodiment of the disclosure, the AFP and prolactin are tested incombination with PP12 to detect fetal membrane rupture.

In another embodiment of the disclosure, PP12 and AFP are used to detectfetal membrane rupture.

It should be noted that PP12 and AFP are believed to be more stable thanmost other suggested biomarkers and are found in higher concentrationsin cervicovaginal secretions than in maternal blood. Consequently, thesetwo markers can be used in combination with each other, or with theother markers.

In another embodiment of the disclosure creatinine and urea are used asmarkers, alone or in combination with other markers.

Another marker that may be used in Interleukin 6 (IL-6), alone or incombination with the markers listed above.

Specifically, monoclonal and polyclonal antibodies are strategicallypositioned at a test site on the lateral flow permeable test strip.

In a further embodiment, preeclampsia may be detected on the samelateral flow strip by using cystatin C, beta2-microglobulin, serumamyloid A, C-reactive protein (CRP), and neopterin.

This may be an argument that the combo PP12 combined with AFP is abetter test for PROM, as they could be more stable over time than justthe one alone. So in addition to the combo approach yielding a higherspecificity for PROM (any of the molecules may be in higherconcentration in the amniotic leaking fluid), one or another of themmaybe more stable and therefore more accurate results if the patientdoes not present to the Doctor when she first starts leaking and thenmaybe stops leaking.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be better understood and objects other than thoseset forth above will become apparent when consideration is given to thefollowing detailed description thereof. Such description makes referenceto the annexed drawings wherein:

FIG. 1 is a perspective view of a swab;

FIG. 2 is a perspective view of a new vial;

FIG. 3 is a front view of the vial;

FIG. 4 is a side view of the vial;

FIG. 5 is a side view of one embodiment of the lateral flow assay;

FIG. 6 is a side view of another embodiment of the lateral flow assay;

FIG. 7 is a side view of yet another embodiment of the lateral flowassay;

FIG. 8 a is a perspective view of a cassette;

FIG. 8 b is a top view of the cassette;

FIG. 9 is an exploded view of the cassette; and

FIG. 10 is a perspective view of the timing device.

DETAILED DESCRIPTION OF THE DISCLOSURE

Referring to FIGS. 1-9, the obtaining a fluid sample is the first stepin the detection for the detection of the PROM or PPROM. A sterile longhandled swab 1 is removed from its packaging. The tip 2 of the swabshould not touch anything prior to its insertion into the vaginal vault.The tip 2 is inserted two to three inches into the vaginal vault. Afteranywhere from about 15 seconds to about three minutes, the swab may bewithdrawn.

A buffer vial 3 shipping cap is opened, and the swab tip 2 is placedinto the vial 3. The buffer in the vial may be a saline or phosphatebuffer 19, or any other non-reactive buffer and preservatives (i.e.,azide, proclin, thimerosal) as deemed necessary. While the vial that isused may be a standard vial, one embodiment of the vial 3 allows for astand alone vial that does not tip over. Specifically, the stand alonevial 3 comprises a vial 30, with a bottom section 31 and a top section32. The top section 32 has an opening 34 and a female collar 35 aroundthe circumference of the opening 34. A stand 36 is attached to thebottom section 31 of the vial.

A male collar 37 snaps into the female collar. The male collar 37comprises a first arm 38 and a second arm 39 positioned opposite to eachother on the male collar 37.

A dropper 40 is positioned at and integral with the distal end 41 of thefirst arm 38. The dropper 40 comprises a stopper 42 having a hole 43therein, said stopper 42 positioned on a top side 44 of the distal end41 of said first arm 38. A dispenser 45 is integral with the stopper.The dispenser 45 is positioned on the underside of the first arm 38. Thestopper 42 positioned so when said first arm 38 is bent, said stopper 42fits snuggly in said opening of said vial, allowing for the dispensingof any liquid in said vial.

A cap 50 is positioned at and integral with the distal end 51 of thesecond arm 39, such that the cap fits snuggly into the opening 34 of thevial 3. The cap 50 at the distal end 51 of the second arm 39 can bepositioned on the top side 52 of the second arm 39. In anotherembodiment, the cap 50 can be positioned on the bottom side 53 of saidsecond arm 39 so that the arm is twisted to fit the cap 50 into theopening 34 of the vial 3.

After the swab tip 2 is placed in the buffer solution 19, the swab stick20, which is scored 21, is broken off at a pre-scored location on theswab, and the vial is gently mixed for about a 15 seconds to 1 minute,with the swab tip in the vial 2. Either before or after mixing the vial,the dropper 9 is fitted into the opening 11 of the vial 3. The solutionmay sit for about 15 seconds to five minutes, as needed.

A lateral flow assay strip positioned in a cassette is removed from afoil pouch, and about three to four drops of sample/buffer solution isadded to the sample well of the cassette.

This lateral flow assay device, such as that used in Ser. No.12/382,570, (filed Mar. 18, 2009, herein incorporated by reference) isused to test for PROM and PPROM by detecting the presence of theantigens in the buffer solution that were collected from the vaginalvault via the swab.

There are a variety of proteins or substances which can be tested on thelateral flow assay device, in a variety of combinations. Substances andproteins which can be tested include (PP12) and (BETA-Ig-H3),alpha-fetoprotein (AFP) prolactin, hCG, fetal fibronectin, etc., andcombinations thereof. Embodiments may include at least two of theantigens listed, to ensure more accurate results. In one embodiment ofthe disclosure both PP12 and alpha-fetoprotein (AFP) are tested.

The lateral flow assay device 22 being used for the detection of theantigens is comprised of a platform strip 24. It is preferred that theplatform strip 24 be impermeable, and, preferably, laminated, and tothat end, a plastic platform strip may be used. The platform strip 24 iselongated. Positioned on the top side of the platform strip 24 is apermeable membrane testing strip 25, where the mixture can bind or stickto the immobilized capture reagent, causing a color reaction, indicatingthe presence of the antigens. The presence and/or amount of analyte inthe sample may be determined by the visibility of a line formed by thecapture reagent 25 a, specific for, in this case, PP12 andalpha-fetoprotein.

There may be more than one capture reagent and thus more thus more thanone capture reagent site when a multiple of analytes are being(possibly) be tested and examined. There may also be a control reagent25 b, which is used to verify that the test is not giving any falsepositives or false negatives (depending on how the control isconstructed). There may also be a second control region 25 c so thatthere may be both a negative and a positive control. In addition, thecontrol line could form a negative sign and the analytes or controlscould form a positive line. The platform strip gives “body” and strengthto the longitudinally positioned testing strip. The permeable membranetesting strip, which could be more properly called a detection membranestrip 25 may be composed of a series of porous material pieces such as,paper, cotton, polyester, glass, nylon, mixed cellulose esters, spunpolyethylene, polysulfones, and the like. Preferably, nitrocellulose,nylon or mixed cellulose esters are used for the analyte detectionmembrane strip 25. It can be attached to the platform strip by anynumber of means, including a variety of simple glues or tape, as long asthe glues do not permeate up through and to the surface of the permeablemembrane testing strip 25.

A sample receiving pad 26 to which the buffered sample is added ispositioned on top of and at a proximal end of the non-permeable platformstrip 24 while in contact with the permeable membrane testing strip 25.More specifically, the distal end 63 of the sample receiving pad 26should either be in physical contact with the proximal end 64 of thepermeable membrane testing strip 25 or the distal end 63 of the samplereceiving pad 26 should be in contact with and overlap the proximal end64 of the sample receiving pad 26. The sample receiving pad 26 may becomposed of a series of porous material pieces such as, paper, cotton,polyester, glass, nylon, mixed cellulose esters, spun polyethylene,polysulfones, and the like. Paper, cotton, polyester, glass fiber,and/or polyethylene are generally used for the sample receiving pad. Aconjugate pad 28 positioned above or below, or even next to the samplepad allows for the tagging of the sample.

The conjugate pad itself 28 is comprised of paper, cotton, polyester,glass fiber, or polyethylene. More importantly than the material of theconjugate pad are the lyophilized colorants in the pad. The dried orlyophilized conjugate in the conjugate membrane 28 may consist of latexmicroparticles, enzymatic, fluorescent, or visually observable tags.These other tags include metal sols, enzymatic, fluorescent, latexmicroparticles, and the like, preferably colloidal gold particles. Theconjugate is to provide a means of identifying any reaction or bindingat the site of the capture reagent 25 a. These means can include visual,fluorescence, radioactive, etc. The colorant is attached to anon-specific mouse monoclonal antibody which binds to an epitope of theantigens in the sample.

Additionally, the monoclonal antibodies to the antigens being tested maybe attached to the gold, latex, selenium, carbon or other metalliccolloids that are used as the colorant. By having a monoclonal antibodyattached to the colorant, there is a greater enhancement of the resultson the test strip as the colorant, with the monoclonal antibody bound tothe specific ligand, migrates to the testing site. Specificity isenhanced, as well as the color at the detection site.

The conjugate pad 28 may be positioned on top or below the samplereceiving pad 26.

In another embodiment a semi-permeable membrane 73 resides directly ontop of the sample receiving pad 26, and the conjugate pad 28 resides ontop of the semi-permeable membrane 73. The semi-permeable membrane 73serves as a temporary obstruction which breaks down when the combinedeffects of the sample volume and surface tension cause the sample to runeventually through semi-permeable membrane laminating membrane 24.Hatched fibers may be used in semi-permeable membrane 73 creatingmicropores. The extended time for the mixing of the of the analyte andthe conjugate results in greater analyte detection sensitivity. Thesemi-permeable membrane is constructed of micropores of diametersbetween 0.01 microns and 20 microns, allowing the sample to slowlydiffuse through the semi-permeable membrane. The semi-permeable membranemay also be constructed of hydrophobic material or hatched fiberscreating micropores, thus allowing for the slow diffusion of samplemixed with conjugate through the semi-permeable membrane. Differentsemi-permeable membranes with different sizes ranges can be used, independing on the biological sample for which the assay is beingperformed. For instance, one could have a semi-permeable membrane havinga preferred pore size range of about 10 about 20 microns. In otherinstances, the sizing of the membrane can be used to screen for certainligands or other materials. For the present disclosure, a semi-permeablemembrane having a pore size range of about 8 to about 20 nm to screen isused test for proteins.

As the now conjugated sample 24 migrates into the flow membrane, theanalyte-particle conjugate is carried along flow path 46 where resolvedon the membrane coated with analyte reactive substances. In a positiveresult, the conjugate-analyte complex reacts with the capture antibodiesattached to the analyte detection membrane/e 25 at site 25 a.

The analytes at the detection site include polyclonal anti-human PP12antibodies and monoclonal and polyclonal anti-human alpha-fetoproteinantibodies. The detection site may also contain monoclonal antibodies,which were mixed with the polyclonal antibodies prior to being dried orlyophilized onto the test strip membrane. The combination of thealpha-fetoprotein and the PP12 has an increased signal due to thecombining of the signals from the target molecules contained in themicrogram/nanogram range in the amniotic fluid. Generally, the ratio ofpolyclonal antibodies blended together are 75% PP12 to 25%alpha-fetoprotein antibodies. In addition, it is known (Shahin et al.)that AFP rises during the 1^(st) and 2^(nd) Trimester and decreasesduring 3^(rd) Trimester while PP12 continues to rise during 3^(rd)Trimester this making the combination for detection for the presence ofamniotic fluid in cervico-vaginal fluid more sensitive throughoutpregnancy.

In another embodiment, polyclonal anti-human antibodies may bepositioned at the site of the colorants, to allow for an increase in thechemical complex at the reaction site.

As noted above variety of different antibodies may be used in differentcombinations to test for PROM. In the case of beta-Ig-H3, there is someevidence that the antigen is found is small amounts in serum. Thus, adilution series may be set up to dilute the vaginal swab sample such fortesting. As the sample is diluted, only the presence of beta-Ig-H3 inthe vaginal sample will be detected.

The monoclonal anti-human PP12 antibodies being used (obtained from R &D Systems) were produced from a hybridoma resulting from the fusion of amouse myeloma with B cells obtained from a mouse immunized with purifiedE coli-derived recombinant human insulin-like growth factor bindingprotein 1 (rhPP12). The IgG fraction of the ascites fluid was purifiedby Protein G affinity chromatography. PP12 is one of several proteinsthat bind IGF with high affinity and modulate IGF activity. PP12 bindsequally well to IF-I and IGF-II. PP12 is expressed in liver, deciduas,kidneys and is the most abundant in amniotic fluid. The antibody islyophilized from a 0.2 um filtered solution in phosphate-buffered saline(PBS) with 5% trehalose. PBS is also used to reconstitute the antibody.If reconstituted with 1 mL of PBS, the antibody concentration will be500 ug/mL.

Polyclonal anti-human PP12 antibodies can be used. More specifically,the use of Human PP12 Affinity Purified Polyclonal antibodies can vastlyreduce the number of false negatives often found in other tests, due tothe attachment or attraction to the number of different epitopes isgreatly increased. Put another way, the polyclonal antibodies bind tomultiple epitopes. This allows for a greater chance of capturing themajority PP12 molecules as they tansverse the membrane The antibody isproduced in goats or sheep immunized with purified E-coli derived,recombinant human insulin-like growth factor binding protein 1 (rhPP12).PP12 specific IgG was purified by human PP12 affinity chromatography.Going through the same lyophilization process as the monoclonalantibody, reconstitution of the lyophilized polyclonal antibody yieldsan antibody concentration of 0.1 mg/ml.

The lateral flow assay test may also test for alpha-fetoprotein. Bothmonoclonal anti-human alpha-fetoprotein antibody and polyclonalanti-human alpha-fetoprotein antibody are used. It should be noted thata number of different monoclonal anti-human PP12 antibodies andanti-human alpha-fetoprotein antibodies are used, wherein the antibodiesare specific for different specific epitopes. This further allows for anincrease in the accuracy of the test.

Other monoclonal and polyclonal antibodies may be used with respect toother antigens that may be used on the test strip for the termination ofPROM. The monoclonal and polyclonal antibodies may be used in thearrangements described above, and manufactured as described or by othermanufacturing techniques used to make monoclonal or polyclonalantibodies.

The polyclonal antibodies positioned aspecific for both thealpha-fetoprotein (why not use AFP everywhere) and the antibodiesspecific for the PP12 may be mixed, or kept in separate lines. In eitherversion of the test, the antibodies in the capture site may be in astraight line, circle, “+” sign, or any other design that will bedistinctive and easy to recognize. Similarly, any and all monoclonalantibodies that may be positioned at the capture site are positioned ina similar fashion.

A control site 25 b is positioned following the capture site 25 a. Thecontrol site gives a positive result in the form of a line, circle, etc,in the presence of the colorant tagged antibodies. If there is nopositive line, the test is faulty, and needs to be repeated. Rabbitantimouse antibodies are used at the control site.

It should be noted that variations of the basic lateral flow devicedescribe above, such as a change in the position of the semi-permeablemembrane may unexpectedly greatly improve the accuracy of the lateralflow assay, and allows for crisp clear reaction lines that allow foreasy visual or machine readability.

In one embodiment, and as shown in FIG. 6, the semi-permeable membrane73 is positioned on top of and in contact with the old conjugate pad 28,and underneath and in contact with and underneath the sample pad 26. Thedistal end 94 of the semi-permeable membrane 73 overlaps onto, or, moreprecisely, on top of the proximal end 64 of the permeable test strip 25.In this circumstance, it is preferred that the semi-permeable membrane73 be in contact with and preferably overlapping the permeable membrane25 at proximal end 64. The conjugate pad 28 and the permeable test strip25 are not in contact with each other.

The semi-permeable membrane 73 serves a couple of different purposeswhen positioned over the gold conjugate pad 28 and underneath the samplepad 26. First, particulate matter and ligands not being tested can betrapped by the semi-permeable membrane 73, dependent of course on thepore size of the semi-permeable membrane 73. This in and of itselfgreatly improves results, as the problem of non-specific binding iseliminated. Second, the rate of the flow of the sample is decreased,thereby preventing the diluent or supernatant from washing over theconjugate pad 28, thereby producing inconclusive or inaccurate results.Third, and as noted above, the semi-permeable membrane 73 isrestrictive, and slows down the flow of the conjugate sample, therebyincreasing the amount of time the conjugate reacts with the ligand oranalyte, giving clearer, more defined, results. By reducing the speed ofthe flow of sample, thereby resulting in the sample and conjugate labelbeing in contact for a longer amount of time before flowing along thesemi-permeable membrane, thereby allowing more time for ligands and goldconjugates to continue to bond.

It should also be noted that the flow of fluid along the lateral flowassay is reduced, which also aids in the reading of the results. Thedetection site 25 a is not overwhelmed by a volume of fluid which mayinadvertently washout some of the antibody or other detection means atsite 25 a.

In another embodiment of the disclosure (FIG. 7), the semi-permeablemembrane 73 is positioned under and in contact with the conjugate pad 28which in turn is positioned under and in contact with the sample pad 26.In this embodiment, the distal end of the semi-permeable membrane 73 isagain in contact with and overlapping the permeable test strip 25 of thelateral flow assay. As described above, neither the conjugate pad northe sample pad are in contact with the permeable test strip. Only thesemi-permeable membrane 73 is in contact with and on top of thepermeable test strip.

The length of the sections of the semi-permeable membrane and the samplereceiving pad overlapping the permeable membrane is normally no morethan about 5-8 millimeters. This distance can be varied depending on theexperiments run, and the preference of the user, if so desired.

While it is generally preferred that gold conjugate particles be used,other metal sol conjugates may also be used. As an alternative to usingmetals (generally attached to antibodies), antibodies attached to a dyewith an extinction coefficient equal to or greater than gold may beused. (The metal sol particles or dyes should have a high extinctioncoefficient equal to or greater than gold.) There are a large number ofother visible conjugates that may be used for the attachment of the tagto a ligand. Indeed, in some circumstances, the conjugate need not bevisible to the naked eye, but must be detectable by some means such asUV light, other light frequencies, readable by machine, etc.

The solid phase particles useful in connection with the disclosure maycomprise, for example, particles of latex or of other support materialssuch as silica, agarose, glass, polyacrylamides, polymethylmethacrylates, carboxylate modified latex and Sepharose. Preferably, theparticles will vary in size from about 0.02 microns to about 10 microns.In particular, useful commercially available materials include 0.99micron carboxylate modified latex, cyanogen bromide activated Sepharosebeads (Sigma), fused silica particles (Ciba Corning, lot #6),isothiocyanate glass (Sigma), Reactogel 25DF (Pierce) andPolybead-carboxylate monodisperse microspheres. In accordance with thedisclosure, such particles may be coated with a layer of FAB, intactantibody, proteins, peptides, lipids, and the like, coupled thereto in amanner known per se in the art to present the solid phase component.

Additionally, in another component of the lateral flow assay, areservoir absorbent pad 27 is positioned on top of and at a distal end65 of the non-permeable membrane platform strip 24 while in contact withthe distal end 65 of the permeable membrane test strip 25. It ispreferred that the proximate end 67 of the reservoir absorbent pad 27overlaps the distal end 66 of the permeable membrane test strip 25,while at the distal end of the reservoir absorbent pad is attached tothe distal end of the non-permeable membrane platform strip. Thereservoir absorbent pad 27 helps draw the fluid sample across thepermeable membrane testing strip 25 by capillary action. The reservoirpad may be composed of a series of porous material pieces such as,paper, cotton, polyester, glass, nylon, mixed cellulose esters, spunpolyethylene, polysulfones, and the like. Preferably, the reservoirabsorbent pad 27 is comprised of paper, cotton, polyester, glass fiber,or polyethylene.

In one embodiment of the disclosure, the assay strip is in a cassette200. The cassette is a case which encompasses a bottom section 201 and atop section 202. The bottom section contains a seat 203 into which thelateral strip 204 fits. The top section 202 contains a sample well(opening) 205 under which is positioned the sample site of the lateralflow assay strip.

Three or four drops of the sample/buffer are added to the sample well205 of the cassette 200. After an appropriate amount of time, theresults of the test are visible through the test window 206. Normally,it is advisable to wait 5-10 minutes for test results to manifest.Strong leakage of amniotic fluid (higher analte concentrations) may makethe results visible early (within 1-3 minutes) while a small leak maketake a full 10 minutes. The test is valid even if the stripes are faintor uneven.

To assist in determining the passage of time once the sample/buffer hasbeen added to the sample well, a timer 207 is included in the cassette200. While electronic or analog timers could be used, the presentdisclosure includes a timer 207 based on the movement of a fluid bycapillary action. The timer 207 being used (created by TimestripTechnical Services Ltd., U.S. Pat. No. 7,232,253, herein incorporated byreference) can be designed to run from about five to about twentyminutes, with an accuracy of +10% in time under isothermal conditions.By pressing the button 300, a seal (not shown) is broken, and fluid 208flows, by capillary action (not shown), to an enclosed cavity 301 forwhich there are markings 302 indicating the amount of time which haspassed since the button 300 was pressed at the time of the applicationof the sample.

The capillary flow timer 207 fits on a raised surface 303 on the bottomsection 201 of the cassette. The top section 202 includes a window 304for the button 300 and a second timer window 305 to view the elapsedtime.

Once the sample is added, and the button is pressed, the user of theassay kit can periodically check to see how much time has elapsed inorder to get an accurate reading of the results and to determine, inpart, the seriousness of the PPROM. At that point, the OB/GYN can make adetermination as to the method of treatment.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the specification is hereby incorporated herein byreference.

While the disclosure has been described in conjunction with specificembodiments, it is evident that many alternatives, modifications andvariations will be apparent to those skilled in the art in light of theforegoing description. Accordingly, the present disclosure attempts toembrace all such alternatives, modifications and variations that fallwithin the spirit and scope of the appended claims.

What is claimed is:
 1. A chromatographic specific binding assay stripdevice for the detection of amniotic fluid in vaginal secretions ofpregnant women due to premature rupture of fetal membranes, saidchromatographic specific binding assay strip device comprising: a) anon-permeable platform strip; b) a permeable membrane testing strippositioned on top of the non-permeable platform strip, said permeablemembrane testing strip further comprising a test site for the detectionof said premature rupture of fetal membranes, said site comprising ablend of at least: i) a first set of polyclonal antibodies for thedetection of a first biomarker, said first biomarker indicating saidpremature rupture of fetal membranes, said first set of polyclonalantibodies being specific for Alpha-Fetoprotein (AFP); and ii) a secondset of polyclonal antibodies for the detection of a second biomarker,said second biomarker being indicative of said premature rupture offetal membranes, said second set of polyclonal antibodies being specificfor Placental Protein 12 (PP-12); iii) a first set of monoclonalantibodies for the detection of said first biomarker said indicatingsaid premature rupture of fetal membranes said first set of monoclonalantibodies being specific for AFP; and iv) a second set of monoclonalantibodies for the detection of second biomarker, said second biomarkerbeing indicative of said premature rupture of fetal membranes saidsecond set of polyclonal antibodies being specific for PP-12; andwherein said first set of polyclonal antibodies and said second set ofpolyclonal antibodies are intermingled with said first set of monoclonalantibodies and said second set of monoclonal antibodies in said blendfor said test site; c) a sample receiving pad positioned on top of andat a proximal end of the non-permeable platform strip while in contactwith a proximal end of the permeable membrane testing strip; d) areservoir pad positioned on top of and at a distal end of thenon-permeable membrane testing strip while in contact with a distal endof said permeable membrane test strip; and e) a conjugate pad positionedon top of or below said sample receiving pad, said conjugate padcomprising a permeable membrane containing a conjugate, said conjugatecomprising a colorant attached to a vehicle which can attach to saidbiomarker.
 2. The chromatographic specific binding assay strip device ofclaim 1, wherein said first set of polyclonal antibodies and said secondset of polyclonal antibodies intermingled with said first set ofmonoclonal antibodies and said second set of monoclonal antibodies insaid blend are positioned at the test site in the shape of a plus sign.3. The chromatographic specific binding assay strip device of claim 1,wherein said blend further comprising at least one biomarker selectedfrom the group consisting of beta-Ig-H3, prolactin and hCG, and fetalfibronectin.
 4. The chromatographic specific binding assay strip deviceof claim 1, further comprising a semi-permeable membrane, wherein saidsemi-permeable membrane is positioned between said sample receiving pad,and said conjugate pad residing on top of said semi-permeable membrane.5. The chromatographic specific binding assay strip device of claim 1,further comprising a semi-permeable membrane, wherein saidsemi-permeable membrane is positioned over said conjugate pad, saidconjugate pad being positioned over said sample receiving pad.
 6. Thechromatographic specific binding assay strip device of claim 1, furthercomprising a cassette, said cassette comprising: a) a bottom section,said bottom section comprising a seat into which the lateral strip fits,and; b) a top section, said top section having the ability to be securedto the bottom section, said top section comprising: i) a sample wellopening into which the sample is added; and ii) a test window whichallows for the viewing of the results of the test, wherein, said topsection and said bottom section are secured together.
 7. Thechromatographic specific binding assay strip device of claim 6, saidcassette of said chromatographic specific binding assay strip devicefurther comprising a timing device, said timing device being positionedin the bottom section of the cassette of said chromatographic specificbinding assay strip device, said top section of said cassette of saidchromatographic device having a button for commencing the timer and atiming window to view elapsed time.
 8. The chromatographic specificbinding assay strip device of claim 7, wherein said timing deviceoperates by means of capillary action.
 9. The chromatographic specificbinding assay strip device of claim 1, further comprise a control siteon said permeable membrane assay strip.
 10. A kit for the detection ofamniotic fluid in vaginal secretions of pregnant women due to prematurerupture of fetal membranes, said kit comprising: a) a chromatographicspecific binding assay strip device for the detection of amniotic fluidin vaginal secretions of pregnant women due to premature rupture offetal membranes, said chromatographic specific binding assay stripdevice comprising: i) a non-permeable platform strip; ii) a permeablemembrane testing strip positioned on top of the non-permeable platformstrip, said permeable membrane testing strip further comprising a testsite for the detection of said premature rupture of fetal membranes,said site comprising a blend, said blend comprising at least: i) a firstset of polyclonal antibodies for the detection of a first biomarker,said first biomarker indicating said premature rupture of fetalmembranes, said first set of polyclonal antibodies being specific forAFP; and ii) a second set of polyclonal antibodies for the detection ofa second biomarker, said second biomarker being indicative of saidpremature rupture of fetal membranes, said second set of polyclonalantibodies being specific for PP-12; iii) a first set of monoclonalantibodies for the detection of said first biomarker said indicatingsaid premature rupture of fetal membranes said first set of monoclonalantibodies being specific for AFP; and iv) a second set of monoclonalantibodies for the detection of second biomarker, said second biomarkerbeing indicative of said premature rupture of fetal membranes saidsecond set of polyclonal antibodies being specific for PP-12; andwherein said first set of polyclonal antibodies and said second set ofpolyclonal antibodies are intermingled with said first set of monoclonalantibodies and said second set of monoclonal antibodies in said blendfor said test site; iii) a sample receiving pad positioned on top of andat a proximal end of the non-permeable platform strip while in contactwith a proximal end of the permeable membrane testing strip; iv) areservoir pad positioned on top of and at a distal end of thenon-permeable membrane testing strip while in contact with a distal endof said permeable membrane test strip; and v) a conjugate pad positionedon top of or below said sample receiving pad, said conjugate padcomprising a permeable membrane containing a conjugate, said conjugatecomprising a colorant attached to a vehicle which can attach to saidbiomarker; and b) a cassette comprising: i) a bottom section, saidbottom section comprising a seat into which lateral strip fits, and; ii)a top section, said top section having the ability to be secured to thebottom section, said top section comprising: i) a sample well openinginto which the sample is added; and ii) a test window which allows forthe viewing of the results of the test, said top section and said bottomsection are secured together; c) at least one vial; d) at least oneswab; and e) a buffer solution.
 11. The kit according to claim 10, saidsite for detecting said premature rupture of fetal membranes, whereinsaid swab is a long handled swab, said long handled swab having: a) asoft absorbent material; and b) and a handle, said handle beingcomprised of either plastic or wood, said soft absorbent materialpositioned on one end of said handle.
 12. The kit according to claim 11,wherein said handle is scored.
 13. The kit according to claim 10,wherein said cassette of said chromatographic specific binding assaystrip device further comprises a timing device, said timing device beingpositioned in the bottom section of the cassette of said chromatographicspecific binding assay strip device, said top section of said cassettehaving a button for commencing the timer and a timing window to viewelapsed time.
 14. The kit according to claim 13, wherein said timerfunctions by capillary action.
 15. The kit according to claim 10,wherein said buffer solution is selected from the group consisting of asaline solution and a phosphate solution.
 16. The kit according to claim10, wherein said vial is a stand alone vial, said stand alone vialcomprising: a) a vial, said vial comprising: i) a bottom section; andii) a top section, said top section comprising: 1) an opening for thevial; and 2) a female collar around said opening; b) a stand attached tosaid bottom section of said vial; c) a collar integral and situatearound the circumference of the open end of said vial; d) a male collarthat snaps into said female collar, said mail collar comprising a firstarm and a second arm positioned opposite to each other on said collar;e) a dropper positioned at and integral with the distal end of the firstarm, said dropper comprising: i) a stopper having a hole therein, saidstopper positioned on a top side of said distal end of said first arm;and ii) a dispenser integral with the stopper positioned on theunderside of said first arm so when said first arm is bent, said stopperfits snuggly in said opening of said vial allowing for the dispensing ofany liquid in said vial; and f) a cap positioned at and integral withthe distal end of the second arm, such that said cap fits snuggly intosaid opening of said vial.
 17. The kit according to claim 16, whereinsaid cap of said distal end of said second arm can be positioned on thetop side of said second arm.
 18. The kit according to claim 16, whereinsaid cap of said distal end of said second arm can be positioned on thebottom side of said second arm.
 19. The kit according to claim 10,wherein said first set of polyclonal antibodies and said second set ofpolyclonal antibodies intermingled with said first set of monoclonalantibodies and said second set of monoclonal antibodies are positionedat the test site in the shape of a plus sign.
 20. The kit according toclaim 10, further comprising at least one other marker selected from thegroup consisting of -beta-Ig-H3, prolactin, hCG, and fetal fibronectinand combinations thereof.
 21. The kit according to claim 20, whereinsaid PP12 biomarkers and alpha-fetoprotein biomarkers are used to detectfetal membrane rupture.
 22. The kit according to claim 10, furthercomprising a semi-permeable membrane, wherein said semi-permeablemembrane is positioned between said sample receiving pad, and saidconjugate pad residing on top of said semi-permeable membrane.
 23. Thekit according to claim 10, further comprising a semi-permeable membrane,wherein said semi-permeable membrane is positioned over said conjugatepad, said conjugate pad being positioned over said sample receiving pad.24. The kit according to claim 10, further comprising a cassette, saidcassette comprising: a) a bottom section, said bottom section comprisinga seat into which the lateral strip fits, and; b) a top section, saidtop section having the ability to be secured to the bottom section; saidtop section comprising: i) a sample well opening into which the sampleis added; and ii) a test window which allows for the viewing of theresults of the test, wherein said top section and said bottom sectionare secured together.
 25. The kit according to claim 23, wherein saidcassette of said chromatographic specific binding assay strip devicefurther comprises a timing device, said timing device being positionedin the bottom section of the cassette of said chromatographic specificbinding assay strip device, said top section of said cassette having abutton for commencing the timer and a timing window to view elapsedtime.
 26. The kit according to claim 25, wherein said timing deviceoperates by means of capillary action.
 27. A stand alone vial, saidstand alone vial comprising: a) a vial, said vial comprising: i) abottom section; and ii) a top section, said top section comprising: 1)an opening for the vial; and 2) a female collar around said opening; b)a stand attached to said bottom section of said vial; c) a collarintegral and situated around the circumference of the open end of saidvial; d) a male collar that snaps into said female collar, said mail,collar comprising a first arm and a second arm positioned opposite toeach other on said collar; e) a dropper positioned at and integral withthe distal end of the first arm, said dropper comprising: i) a stopperhaving a hole therein, said stopper positioned on a top side of saiddistal end of said first arm, and ii) a dispenser integral with thestopper, said dispenser positioned on the underside of said first arm,said stopper positioned so when said first arm is bent, said stopperfits snuggly in said opening of said vial, allowing for the dispensingof any liquid in said vial; and f) a cap positioned at and integral withthe distal end of the second arm, such that said cap fits snuggly intosaid opening of said vial.
 28. The stand alone vial according to claim27, wherein said cap of said distal end of said second arm can bepositioned on the top side of said second arm.
 29. The stand alone vialaccording to claim 27, wherein said cap of said distal end of saidsecond arm can be positioned on the bottom side of said second arm.